Dear Skyline Users,

The Skyline Team is excited to give you Skyline 21.1, the product of another 7 months of effort in response to your feedback and use of Skyline.

Over the past 7 months, you have started 11,000 instances (on average) of Skyline or Skyline-daily each week, and downloaded the software 1400 times per month. There have been 1000 posts to the support board. You showed your enthusiasm for Skyline Online twice, and the May Institute at NEU where some of you got a sneak peak at this release using Skyline-daily.

Improvements in Skyline 21.1 include:

• New! DIA-Umpire integration with library-free DIA data processing. (thanks to the Nesvizhskii lab) - [tutorial]
• New! Skyline Batch for large-scale batch processing of data analysis with Skyline [documentation]
• New! Class Discovery tools (hierarchical clustering and PCA plot) - [documentation]
• Extended crosslinked peptide target support to allow any number of peptides and any number of links, especially important to monitoring disulfide bonds on antibody drug conjugates.
• New UI for direct pasting or File > Import > Tranition List, allowing users to assign meaning to columns.
• Improved Import > Transition List or Assay Library format detection to do a better job of detecting the list and decimal separators regardless of system number format settings.
• Detection plot improvements.
• New spectrum annotation button on the Full-Scan graph to allow full peptide fragment annotation on extraction spectra.
• Added update progress display to RT > Regression and Scheduling plots.
• Added calculated concentration y-axis on the peak area graph, found under right-click > Normalize To.
• Added iRT support for command-line import peptide search.
• Added View > Targets menu item to clarify how to get Targets back after closing, especially for small molecules.
• Improved File > Share to avoid saving on unmodified documents and save directly to the .sky.zip file to avoid overwriting the file on disk for modified documents.
• Enabled the --ui argument for SkylineCmd.exe to show the UI and allow changes that get stored in its user settings file.
• Adding small molecules from View > Spectral Libraries where the library contains molecule list information will preserve the molecule list name.
• Added optional display of ion mobility information in chromatogram graphs.
• File > Export > Spectral Libraries now preserves protein and molecule list name information in the resulting .blib file.
• Extended spectral library support:
  • Support for the latest DIA-NN speclib format.
  • Support for reading MSP-formatted spectral libraries as exported by Thermo's mzVault Viewer.
  • Support library building with .mzid.gz files and use it for MS Amanda DDA search in Skyline.
  • Support library building for invK0 attribute in pepXML written by PEAKS export.
  • Improved error message when a supported spectral library type (.msp, .sptxt, .hlf) is added as the file to use in building a library.
  • Added support for "RTINSECONDS" retention time encoding in an MSP spectral library file comment.
  • Improved error handling when trying to read a damaged BiblioSpec file.
• Improved memory consumption during full-scan data import.
• Added "Simple Ratios" checkbox to Peptide/Molecule Settings > Quantification settings.
• Added new report fields:
  • Added TransitionResult.CycleTimeAcrossPeak report field.
  • Added Protein.ProteinSequenceCoverage report field with the percent of protein sequence amino acids contained in any of the peptides.
  • Added "Library Ion Mobility" report fields.
  • Added "TransitionResultIsQuantitative" and "TransitionResultIsMs1" report fields for MSstats.
• Updated instrument vendor integration:
  • Updated Agilent method export format.
  • Updated the SCIEX WIFF2 DLLs.
  • Updated Waters MassLynx DLLs to 4.8 which returns profile IMS data with flanking zero points.
• Ion mobility improvements found testing Thermo FAIMS support:
  • Negative CoV values now allowed.
  • On export of transition lists, CoV value now pulled from ion mobility library as needed.
  • Ion mobility libraries now support molecules described only as name+mass (was assuming a chemical formula would be available)
  • Viewing raw scan data now shows the scan's ion mobility value when available.
  • Populating an ion mobility library via "Use Results" works properly when two molecules shared the same name but different details
• Other ion mobility UI fixes:
  • Fixed "Transition Settings > Ion Mobility > Use Spectral Library Ion Mobilities" checkbox to avoid state where it would not uncheck.
  • Full Scan graph now redraws on data reimport when showing ion mobility filter information, to ensure correct position of the purple IM filter band.
• Fixed an issue with the Full-Scan view showing only one chromatogram type (MS1 or MS/MS) when both are available.
• Fixed Edit Annotation form to display an error for a value list with not values specified.
• Fixed opening or creating an ion mobility library file before the "Name" field is completed, an error pops up.
• Fixed Import Peptide Search for PRM requiring MS1 filtering settings.
• Fixed problem in number of methods created when exporting an isolation list or method for Thermo Fusion machines.
• Fixed "Ratio to Heavy" to show up even if Heavy is not an internal standard type.
• Fixed support for non-Unimod mods and terminal mods in DDA search with MS Amanda.
• Fixed DDA search with MSAmanda to ignore FileNotFound/DirectoryNotFound when deleting temp files.
• Fixed import peak boundaries into multi-sample wiff files.
• Fixed pepXML spectral library building to accept search results from "peaks_db" as well as "peaksdb".
• Fixed DDA search with MS Amanda fragment tolerance always using precursor tolerance units.
• Fixed NullReferenceException pushing "Add" button from Result File Rules Edit List form.
• Fixed issue with Refine >Associate Proteins with a nameless FASTA sequence.
• Fixed unexpected error typing ')' into the empty element at the bottom of the Targets view.
• Fixed MSFragger spectral library builder to prefer spectra from _uncalibrated.mgf to avoid de-charged fragment ion peaks.
• Improved error message loading .skyd file when it is found to be corrupted.
• Improved audit log hash calculation to avoid the possibility of duplication.
• Fixed bugs with Turkish language settings mostly related to case insesitive pattern matching.
• Fixed library build treating Mascot fixed terminal mods as not overridden by variable terminal mods.
• Fixed an unexpected error when importing results with an ion mobility library when a precursor ion appears more than once in a document.
• Fixed Waters "Unknown Generic Error" due to calling GetDriftTime() on functions existing in Waters .RAW but not listed in _extern.inf.
• Fixed precursor ion spectrum support for WIFF files (use <product> element instead of <precursor>).
• Fixed unexpected error when user specifies a DDA search in the import wizard but then uses the "Back" button to start over.
• Improved error handling when importing small molecule transition lists with inconsistent molecule descriptions.
• Fixed unexpected error exporting method for Bruker timsTOF.
• Fixed bug where Protein Abundance was always #N/A if normalization method is Ratio to Heavy.
• Fixed a bug in the small molecule UI where inappropriate text was having "Peptide" replaced with "Molecule".
• Fixed issues in handling loss-only modifications like water and ammonia:
  • Highlighting in the Targets view for loss-only modifications.
  • Not possible to specify loss-only modifications with Edit > Modify Peptide.
  • Importing Assay Libraries with loss-only modifications on peptides requiring explicit modifcations caused an error.
• Fixed to force choosing a filename with the same extension when a library file is missing.
• Fixed perf and error problems with "Remove Peak" on the Document Grid.
• Fixed irtdb docxml bug.
• Fixed DIA isolation scheme in Import Peptide Search to use the one currently set.
• Fixed Import Peptide Search case with no results files used to create a template document for Skyline Batch.
• Fixed to include PeptideSettings - Filter tab settings in Library Explorer - Add All button.
• Fixed NullReferenceException displaying tooltip on Fold Change Volcano plot if document has small molecules.
• Fixed library build from MaxQuant search results not applying terminal label-type mods.
• Fixed custom formats on columns in the Document Grid would be forgotten as soon as the data was refreshed.
• Fixed Mascot variable mod override logic in library build.
• Fixed TIC Normalization on the Peak Area Replicate Comparison graph.
• Fixed message for opening .sky file inside a ZIP file to work when the file is in a subdirectory.
• Fixed bug preventing DDA search with multiple files of the same name.
• Fixed "An item with the same key has already been added" exception changing Transition Settings in a document that has a molecule with multiple precursors that are the same as each other.
• Fixed problem where DDA search incorrectly adds isotope modifications to the document as structural modifications.
• Fixed problem where rdotp would not appear in the Targets tree if any of the heavy transitions had zero area.
• Fixed problem where "Accuracy" is miscalculated if the Sample Dilution Factor is not 1.
• Fixed unexpected error if using "Modify" menu item on a molecule and changing its chemical formula to result in some precursor transitions no longer existing in the mass distribution.
• Fixed problem where non-quantitative transitions would not show up in Peak Area - Replicates view.
• Fixed highlighting in red "Include all matching scans" if the Acquisition Method is "Targeted" when that is the right option.
• Improved agreement between command-line error logging and non-zero exit codes.
  • Users running Chinese and Japanese language Skyline command-line should update SkylineRunner.exe
• Fixed Tools > Options - Miscellaneous - Clear All Saved Settings for teaching and testing.
• Fixed a Minimize Results error if it was opened while the document was still loading.
• Fixed a sizing layout issue in the FASTA page of the File > Import > Peptide Search wizard.
• Fixed unexpected errors in the RT scheduling graph.
• Fixed Library Match title to contain library name again - broken when Prosit was added.
• Fixed --report-invariant command-line argument to always use a comma field separator.
• Fixed Import Peptide Search to add RT predictor in if adding existing library with iRT values.
• Fixed Import Peptide Search to always show Match Modifications page.
• Fixed notification of iRT calculator modification when using BLIB file.

This release may require a manual upgrade. We hope you'll take the time to visit the Skyline installation page:

https://skyline.ms/skyline64.url

And install this latest release. It should be worth the effort.

(Please note: We are discontinuing 32-bit Skyline. This upgrade requires a 64-bit computer.)

Thanks for your continued use, feedback, and support of the Skyline Project.

Brendan MacLean
Skyline Principal Developer